2,2′-Bipyridinebutyldithiocarbamatoplatinum(II) and palladium(II) complexes: Synthesis, characterization, cytotoxicity, and rich DNA-binding studies

Article abstract of DOI:10.1016/j.bmc.2008.08.021

Butyldithiocarbamate sodium salt (Bu-dtcNa) and its two complexes, [M(bpy)(Bu-dtc)]NO₃ (M = Pt(II) or Pd(II) and bpy = 2,2′-bipyridine), have been synthesized and characterized on the basis of elemental analysis, molar conductivities, IR, ¹H NMR, and UV-vis spectra. In these complexes, the dithiocarbamato ligand coordinates to Pt(II) or Pd(II) center as bidentate with two sulfur atoms. These complexes show 50% cytotoxic concentration (Cc₅₀) values against chronic myelogenous leukemia cell line, K562, much lower than that of cisplatin. The interaction of these complexes with calf thymus DNA was extensively investigated by a variety of spectroscopic techniques. These studies showed that both complexes presumably intercalate in DNA. UV-vis studies imply that they cooperatively bind with DNA and unexpectedly denature the DNA at very low concentrations (～100 μL). Palladium complex breaks the DNA into two unequal fragments and binds stronger to the lighter fragment than to the heavier one. In the interaction studies between the Pt(II) and Pd(II) complexes with DNA, several binding and thermodynamic parameters have been determined, which may provide deeper insights into the mechanism of action of these types of complexes with nucleic acids.

Full text of DOI:10.1016/j.bmc.2008.08.021

Bioorganic & Medicinal Chemistry 16 (2008) 9616–9625
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
0
2
,2 -Bipyridinebutyldithiocarbamatoplatinum(II) and palladium(II) complexes:
Synthesis, characterization, cytotoxicity, and rich DNA-binding studies
a,*
a
b
b
Hassan Mansouri-Torshizi , Mahboube I-Moghaddam , Adeleh Divsalar , Ali-Akbar Saboury
a
Department of Chemistry, University of Sistan & Baluchestan, Daneshgah Street, Zahedan, Iran
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Butyldithiocarbamate sodium salt (Bu-dtcNa) and its two complexes, [M(bpy)(Bu-dtc)]NO (M = Pt(II) or
3
Received 17 June 2008
Revised 2 August 2008
Accepted 5 August 2008
Available online 9 August 2008
0
Pd(II) and bpy = 2,2 -bipyridine), have been synthesized and characterized on the basis of elemental anal-
1
ysis, molar conductivities, IR, H NMR, and UV–vis spectra. In these complexes, the dithiocarbamato
ligand coordinates to Pt(II) or Pd(II) center as bidentate with two sulfur atoms. These complexes show
50% cytotoxic concentration (Cc50) values against chronic myelogenous leukemia cell line, K562, much
lower than that of cisplatin. The interaction of these complexes with calf thymus DNA was extensively
investigated by a variety of spectroscopic techniques. These studies showed that both complexes pre-
sumably intercalate in DNA. UV–vis studies imply that they cooperatively bind with DNA and unexpect-
Keywords:
DNA binding
Cytotoxicity
Platinum complex
Palladium complex
edly denature the DNA at very low concentrations (ꢀ100
lL). Palladium complex breaks the DNA into
two unequal fragments and binds stronger to the lighter fragment than to the heavier one. In the inter-
action studies between the Pt(II) and Pd(II) complexes with DNA, several binding and thermodynamic
parameters have been determined, which may provide deeper insights into the mechanism of action
of these types of complexes with nucleic acids.
Ó 2008 Published by Elsevier Ltd.
1
. Introduction
tack of enzyme-thiol to the metal center and protects normal
tissue without inhibiting the anti-cancer activities of metal dithio-
carbamate complexes.
1
2
Despite the clinical anti-cancer utility of cisplatin, carboplatin,
oxaliplatin, nedaplatin, iproplatin, and several other complexes in
clinical trials, there is a continued interest in the design of new
platinum complexes that shows anti-tumor activities equivalent
or better than these agents.1 Cisplatin is an important cytotoxic
Recently, palladium(II) complexes have attracted significant
2
attention due to their biological activities. Moreover, palladium
1
3
complexes having chelating ligands such as N–N-diamines,
–4
14
15
16
O–S-donor, N–S-amino-thioether, diaminoacids, dicarboxylic
5
17
12,18
drug used in the treatment of a variety of human cancers. It has
acids, and the most important dithiocarbamates
are the re-
some dose limiting toxic side effects such as neurotoxicity, ototox-
cent advances of palladium(II) complexes. It has been assumed
that chelating ligands not only impose the cis coordination of the
palladium(II) complexes but also prevent the formation variety of
aquo, hydroxo, or even polynuclear species which may be formed
from dichloro complexes at physiological pH and ionic strength.
Moreover, such palladium(II) complexes can serve as good models
for the understanding of more inert platinum(II) anti-cancer
drugs. Also, it has been suggested by Saddler et al. that the
palladium complexes may be useful for the treatment of tumors
of the gastrointestinal region where cisplatin fails.
6
icity, and tissue toxicity. Also, there is less nausea, nephrotoxicity,
gastrointestinal, and bone marrow toxicity.7 Moreover, resistance
,8
9
after continued treatment by several mechanisms is witnessed.
Many efforts have been made to reduce the toxicity of platinum
anti-cancer complexes. One of them is using a variety of sulfur-
containing ligands as detoxicant agents against metal-containing
1
0
19
20
drugs. It has been observed that the use of diethyldithiocarba-
mate in combination with cisplatin has protected a variety of ani-
mal species from renal, gastrointestinal and bone marrow toxicity,
induced by cisplatin.1 This may be due to strong binding of plati-
num with dithiocarbamate, which prevents or at least limits the
reaction with other sulfur-containing renal proteins.10 That is
why the chelating sulfur atoms of dithiocarbamates to the plati-
num or palladium moiety selectively inhibits the nucleophilic at-
1
It has been generally accepted that the ultimate target for plat-
inum and palladium anti-cancer agents is likely to be DNA. But the
reaction mechanism of cisplatin by which it induces cell death is
still to be verified. However, cisplatin inhibits the biosynthesis of
DNA and evidences of its in vivo activity at the transcription level
2
1
had been reported. Thus, the cytotoxicity of platinum-based
drugs is perhaps due to the combined effects of the various lesions
*
Corresponding author. Tel.: +98 541 2449807; fax: +98 541 2446565.
2
2,23
E-mail address: hmtorshizi@hamoon.usb.ac.ir (H. Mansouri-Torshizi).
caused by these agents.
0
968-0896/$ - see front matter Ó 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.08.021

H. Mansouri-Torshizi et al. / Bioorg. Med. Chem. 16 (2008) 9616–9625
9617
Keeping the above criteria in mind and considering the point
that the lack of water solubility of some of platinum or palladium
anti-tumor species explains their limited bioavailability and low in
the m(N–CSS) mode has shifted to higher frequencies upon coordi-
nation. This indicates that the nitrogen–carbon double bond char-
acter has increased due to the electron delocalization toward the
platinum or palladium centers. Thus, the above butyldithiocarba-
mate ligand coordinates to Pt(II) or Pd(II) through sulfur atoms.
The second interesting feature in the IR spectra of the Bu-dtcNa li-
gand and its metal complexes is the presence of a single strong
2
4
vivo activity, we synthesized and characterized the two water
soluble and structurally related platinum(II) and palladium(II)
0
dithiocarbamate complexes. Presence of 2,2 -bipyridine as a carrier
ligand in the structure of these compounds which has primary
effect on their anti-tumor properties25 make them to have (i) a
UV–vis absorption band in a region where DNA does not show
absorption and can be monitored in the processes of interaction
studies with DNA and (ii) provide a planar moiety to the complexes
which can intercalate in DNA. Fluorescence titration, electronic
absorption titration, and CD spectroscopy were all employed to
study the interaction of prepared compounds with calf thymus
DNA. In these interaction studies, binding parameters, thermody-
namic parameters, and the types of bindings between these agents
and DNA are also described. Information obtained from these stud-
ies will be helpful to understand the mechanisms of the interaction
between the dithiocarbamate complexes and nucleic acids. These
interaction mechanisms must be quite different from that of
cisplatin.
ꢁ1
ꢁ1
band at 923 cm for Bu-dtcNa and at 1021 and 1026 cm for
[Pt(bpy)(Bu-dtc)]NO
band is attributed to
3
and [Pd(bpy)(Bu-dtc)]NO
3
, respectively. This
3
1
m(SCS) mode which is indicative of symmet-
rical bonding of dithiocarbamate ligand, acting in a bidentate mode
in our complexes (Scheme 1). Otherwise a doublet would be ex-
ꢁ1
pected in the 1000 ± 70 cm region, which indicates an asymmet-
rically bonded ligand or a monodentate bound ligand.32
2.1.2. 1H NMR spectra
The 1H NMR spectral data of butyldithiocarbamate ligand and
0
its corresponding 2,2 -bipyridine platinum(II) and palladium(II)
6
complexes in DMSO-d are summarized in Table 1. The proposed
structure and NMR numbering scheme are given in Scheme 1. In
1
the H NMR spectrum of Bu-dtcNa, the triplet observed at d 0.82,
three multiplets at d 1.21, d 1.39, and d 3.31, and a broad singlet
at d 8.09 are assigned to H-a, H-b, H-c, H-d, and H-e protons,
respectively (see Scheme 1 and Table 1). The signal due to the H-
2
2
2
. Results and discussion
e protons in the spectra of [Pt(bpy)(Bu-dtc)]NO
Bu-dtc)]NO complexes appears at d 11.50 and d 11.51, respec-
tively. The downfield shift of 3.41 ppm in [Pt(bpy)(Bu-dtc)]NO
and 3.42 ppm in [Pd(bpy)(Bu-dtc)]NO as compared to the free li-
3
and [Pd(bpy)
.1. Characterization of compounds
(
3
.1.1. Infrared spectra
The IR spectra of the above-mentioned ligand and complexes
3
3
ꢁ
1
gand indicates the bidentate coordination of Bu-dtcNa ligand to
Pt(II) and Pd(II), and is consistent with the IR spectral data (vide
supra).
have been recorded in the range 4000 to 400 cm as KBr pellets.
Two important bands are of interest: The Bu-dtcNa ligand showed
ꢁ
6–28
1
a strong absorption at 1480 cm . This band, which is assigned to
the N–CSS stretching mode,2
in the IR spectra of [Pt(bpy)(Bu-dtc)]NO
dtc)]NO , respectively. These data suggested that the N–CSS bond
order is between a single bond (
appears at 1525 and 1535 cm
and [Pd(bpy)(Bu-
ꢁ1
The coordination of the dithiocarbamate ligand to platinum or
palladium invariably produces a downfield shift in the position of
its proton signals relative to those of the free dithiocarbamate li-
gand. The extent of this downfield shift decreases regularly with
3
3
ꢁ
1
m
= 1250–1350 cm ) and a double
3
3
ꢁ
1 29,30
distance from the coordination site. This is due to the electron
bond (
m
= 1640–1690 cm ).
As evident from IR spectral data of
ꢁ
donation by –CSS group to palladium or platinum. However, the
the free dithiocarbamate ligand and the corresponding complexes,
0
0
signals corresponding to the H-6,6 (8.67 ppm) and H-5,5
0
(
7.72 ppm) protons of the 2,2 -bipyridine moiety in [Pt(bpy)Cl
2
]
a
b
c
d
CH
e
are invariably shifted upfield in the dithiocarbamate complexes
CH3
CH
2
CH
2
2
NH CSSNa
12
(see Table 1). This may be explained in terms of back donation
0
(I)
of electron density from platinum or palladium to 2,2 -bipyridine
ligand as well as stronger binding of dithiocarbamate to Pt(II) or
5
Pd(II) relative to the chloride anion in [Pt(bpy)Cl
] or [Pd(bpy)Cl
2
].
2
0
4
The protons of 2,2 -bipyridine in [Pt(bpy)(Bu-dtc)]NO
appear as
6
3
S
S
four multiplets centered at 8.67, 8.46, 8.42, and 7.72 ppm, which
a
b
c
d
e
N
3
0
0
0
0
CH3 CH2 CH
CH2 NH
C
M
NO3
are assigned to H-6,6 , H-4,4 , H-3,3 , and H-5,5 protons, respec-
2
0
3'
4'
tively. Similar assignments have been done for protons of 2,2 -
N
(II)
3
bipyridine in the analogous complex [Pd(bpy)(Bu-dtc)]NO (see
6'
Scheme 1 and Table 1). Thus, based on the spectroscopic data,
the structures, which are shown in Figure 1(II) have been assigned
to these two complexes which are also in accord with observed
5'
M=Pd(II) or Pt(II)
2
ꢁ1
ꢁ1
molar conductance values of 101 and 96 cm
X
cm
for
Scheme 1. Proposed structures and nmr numbering schemes of (I) Bu-dtcNa, (II)
M(bpy)(Bu-dtc)]NO
[
Pt(bpy)(Bu-dtc)]NO
3
and [Pd(bpy)(Bu-dtc)]NO , respectively, as
3
[
3
.
Table 1
1
H NMR spectral data of Bu-NH-CSSNa ligand and [M(bpy)(Bu-NH-CSS)]NO
3
complexes in deuterated DMSO
0
Compound
Dithiocarbamate protons
2,2 -Bipyridine protons
0
0
0
0
dH-5,5
dH-a
dH-b
dH-c
dH-d
dH-e
dH-6,6
dH-4,4
dH-3,3
a
b
Bu-dtcNa
0.82 (t)
0.91(t)
1.21(m)
1.34(m)
1.29(m)
1.39(m)
1.61(m)
1.55(m)
3.31(m)
3.46(m)
3.37(m)
8.09(sb)
11.50(sb)
11.51(sb)
—
—
—
—
[
Pt(bpy)(Bu-dtc)]NO
3
8.67(m)
8.50(m)
8.46(m)
8.25(m)
8.42(m)
8.06(m)
7.72(m)
7.64(m)
[
Pd(bpy)(Bu-dtc)]NO
3
0.86(m)
a
Chemical shifts in ppm.
b
s, d, t, q, m, and sb are singlet, doublet, triplet, quartet, multiplet, and singlet broad, respectively.

9
618
H. Mansouri-Torshizi et al. / Bioorg. Med. Chem. 16 (2008) 9616–9625
gand. Bands II, III, IV, and V in platinum complex may be due to
1
20
*
0
first, second, and higher internal
p
?
p
transitions of 2,2 -bipyri-
1
2,35
dine ligand.
also overlapping components of
mate ligand. The above electronic absorption data suggest that
Bands III, IV, and V in the platinum complex have
*
p
? p transitions of dithiocarba-
9
0
0
0
3
7
these complexes have square planar configuration. In palladium
complex, bands II and III show blue shifts by 13–14 nm, respec-
tively, on going from less polar chloroform to more polar dimeth-
ylsulfoxide. Therefore, these bands can tentatively be assigned to
6
0
charge transfer from palladium to 2,2 -bipyridine ligand. Bands
3
*
IV and V are assigned to first and higher internal
p
?
p
transition
0
35
of 2,2 -bipyridine. Also, bands III, IV, and V have overlapping
*
components of
p ? p transitions of dithiocarbamate ligand.
0
0
40
80
120
160
200
2
.2. Cytotoxicity measurements of the Pd(II) and Pt(II)
[complex] (μM)
complexes
Figure 1. The growth suppression activity of the Pd(II) complex (e) and Pt(II)
complex (Ç) on K562 cell line was assessed using MTT assay as described in
material and methods. The tumor cells were incubated with varying concentrations
of the complexes for 24 h.
The prepared platinum and palladium complexes were tested in
vitro against human cancer cell line K562. In this study, various
concentrations of Pd(II) and Pt(II) complexes ranging from 0 to
38
2
50
for 24 h (Fig. 1). The 50% cytotoxic concentration (Cc50) of each
compound was determined 14.3 and 18 M for Pt(II) and Pd(II)
lM of stock solutions were used to culture the tumor cell lines
1
:1 electrolyte.34 Further support for the proposed structures
l
comes from the ratio of the integrated areas under the peaks of
2
0
complexes, respectively (see Fig. 1). As shown in Figure 1, cell
growing after 24 h was significantly reduced in the presence of var-
ious concentrations of the two compounds. Based on this figure, it
is also clear that the Pd(II) and Pt(II) complexes produced a dose–
response suppression on growing of K562 leukemia cell lines. Fur-
thermore, the Cc50 cytotoxicity values of the complexes were
compared to that found for anti-cancer agents used nowadays, that
is, cisplatin under the same experimental conditions. This value
,2 -bipyridine and dithiocarbamate protons being 8:10 for
[
Pt(bpy)(Bu-dtc)]NO
ly, no changes were observed in the H NMR spectra of the above
complexes dissolved in DMSO-d and recorded after 24 h, suggest-
ing no dissociation of dithiocarbamate anions.
3 3
and [Pd(bpy)(Bu-dtc)]NO complexes. Final-
1
6
2
.1.3. Electronic spectra
The maxima absorption bands in electronic absorption spectra
(
154
lM) is much higher as compared to those achieved for the
of above platinum(II) and palladium(II) complexes in distilled
18
metal complexes reported in this article.
water with their extinction coefficients are given in Table 2 and
assigned as follows: band I in the platinum complex is assigned
to MLCT because it is blue shifted by 10–15 nm on going from ace-
tone to water. In this complex, band I may be due to charge
transfer from platinum to
band I may as well be due to the charge transfer from platinum
to
transition is not observed in 2,2 -bipyridine analogous complexes
reported earlier, because it may be hidden in much stronger
2
.3. DNA-binding studies
3
5
*
0
2.3.1. Absorbance titration experiments
p of 2,2 -bipyridine ligand. However,
A fixed amount of each metal complex (0.05 mL of 0.1 mmol/L
stock) was titrated with increasing concentration of DNA (0.2–
0.6 mL for Pt(II) system and 0.02–0.2 mL for Pd(II) system of
0.1 mmol/L stocks) in total volume of 2 mL at 27 °C and 37 °C, sep-
*
36
p
of dithiocarbamate. However, this type of charge transfer
0
1
2
*
0
arately. The values of DAmax, change in the absorbance when all
charge transfer transition from platinum to
p of 2,2 -bipyridine li-
Table 2
3 3
Electronic absorption bands and molar conductance values of [Pt(bpy)(Bu-dtc)]NO and [Pd(bpy)(Bu-dtc)]NO complexes in water
Molar conductance of 1 ꢂ 10ꢁ solution (cm2
4
X
ꢁ1
mol )
ꢁ1
Compound
Band maxima in nm
Band III
Band I
Band II
Band IV
Band V
[
[
Pt(bpy)(Bu-dtc)]NO
Pd(bpy)(Bu-dtc)]NO
3
364(3.15)
__
321(6.74)a
317(2.19)
310(6.34)
307(1.96)
283(1.22)
248(4.71)
208(2.42)
203(2.76)
101
96
3
a
Extinction coefficients in L mol cm ꢂ 10ꢁ4 are given in parentheses.
ꢁ1
ꢁ1
Table 3
Values of
D
A
max and binding parameters in the Hill equation for interaction between Pt(II) and Pd(II) complexes and DNA in 10 mmol/L Tris–HCl buffer and pH 7.0
a
gb
Kc (mol/L)ꢁ1
nd
Errore
Compound
Temperature (°C)
DA
max
[
Pt(bpy)(Bu-dtc)]NO
3
27
0.102
0.222
8
8
0.057
0.036
3.14
2.75
0.012
0.008
3
7
[
Pd(bpy)(Bu-dtc)]NO
3
27
0.047
0.033
8
8
0.317
0.401
1.48
1.79
0.005
0.008
3
7
a
Change in the absorbance when all the binding sites on DNA were occupied by metal complex.
The number of binding sites per 1000 nucleotides.
The apparent binding constant.
The Hill coefficient (as a criterion of cooperativity).
Maximum error between theoretical and experimental values of m
ꢀ.
b
c
d
e

H. Mansouri-Torshizi et al. / Bioorg. Med. Chem. 16 (2008) 9616–9625
9619
retical values of these parameters could be deduced. The results
are tabulated in Table 3, which are comparable with those of
1
1
1
1
60
40
20
00
35
2
7 °C
7 °C
3
2
2
1
1
0
5
0
5
0
3
0
2,2 -bipyridine-platinum and -palladium complexes of dithiocar-
1
2
bamate as reported earlier. The maximum errors between exper-
imental and theoretical values of
ꢀ are also shown in Table 3 which
are quite low. The K, apparent binding constant in the interaction
of [Pd(bpy)(Bu-dtc)]NO with DNA is higher than that of [Pt(bpy)
(Bu-dtc)]NO with DNA (see Table 3). This indicates that the inter-
m
0
500
1000
3
1
/[DNA](mM)-1
8
6
4
2
0
0
0
0
0
3
action affinity of Pd(II) complex to DNA is more than Pt(II) com-
5
plex. Because palladium complexes are about 10 times more
2
0
labile than their platinum analogs. In addition, the data in Table
show that the values of n, the Hill coefficient (as a criterion of
.
2
7 °C
3
.
37 °C
cooperativity), for palladium complex are higher than that of plat-
inum analog. Similar trends are observed in the results of cytotoxic
studies of these two compounds. Knowing the experimental (dots)
and theoretical (lines) values of
imposibility of them on each other, these values of
versus the values of Ln [L] . The results are sigmoidal curves and
m
ꢀ in the Scatchard plots and super-
ꢀ were plotted
20
40
60
80
100
120
m
1
/[DNA] (mM)-1
f
are shown in Figure 4 at 27 and 37 °C. These plots indicate positive
cooperative binding at both temperatures for both complexes.
Finding the area under the above plots of binding isotherms and
Figure 2. The changes in the absorbance of fixed amount of each metal complex in
the interaction with varying amount of DNA at 27 and 37 °C. The linear plot of the
reciprocal of
3
DA versus the reciprocal of [DNA] for [Pt(bpy)(Bu-dtc)]NO . Inset, for
[
Pd(bpy)(Bu-dtc)]NO
3
.
16
using Wyman-Jons equation, we can calculate the Kapp and
ꢄ
D
G°
the values of
for [Pt(bpy)(Bu-dtc)]NO
at 27 °C. Deflections are observed in both plots. These deflections
indicate that at particular [L] , there is a sudden change in enthalpy
b
at 27 and 37 °C for each particular
m
f
and also
D
H . Plots of
b
ꢄ
DH
versus the values of [L]
are shown in Figure 5
b
binding sites on DNA were occupied by metal complex, are given in
Table 3 and Figure 2. These values were used to calculate the con-
3
and the inset for [Pd(bpy)(Bu-dtc)]NO
3
b
centration of metal complex bound to DNA, [L] , and the concen-
f
tration of free metal complex, [L] and
f
m
ꢀ, the ratio of the
of binding which may be due to binding of metal complex to mac-
romolecule or macromolecule denaturation.
concentration of bound metal complex to total [DNA] in the next
experiment, that is, titration of fixed amount of DNA (0.05 mL of
0
(
.1 mmol/L stock) with varying amounts of each metal complex
0.1–0.18 mL for Pt(II) system and 0.025–0.25 mL for Pd(II) system
of 0.1 mmol/L stock) in total volume of 2 mL at 27 and 37 °C, sep-
arately. Using these data ( ), the Scatchard plots were con-
ꢀ and [L]
2
.3.2. Analysis of denaturation data and evaluation of
thermodynamic parameters
The above platinum(II) and palladium(II) complexes can dena-
ture DNA. In this experiment, the sample cell was filled with
m
f
structed for the interaction of each metal complex at two
temperatures of 27 and 37 °C. The Scatchard plots are shown inFig-
1
.8 mL DNA (0.1 mmol/L of Pt system and 0.04 mmol/L of Pd sys-
tem). In these concentrations, the absorption of DNA is around
.8 and 0.4, respectively. However, reference cell is only filled with
1.8 mL Tris–HCl buffer. Both cells were set separately at constant
temperature of 27 or 37 °C, and then 25 L solution of metal com-
ure
Bu-dtc)]NO
gesting cooperative binding. To obtain the binding parameters,
the above experimental data ([L]
ꢀ) were substituted in Hill equa-
ꢀ ¼ gðK½Lꢃ Þ =ð1 þ ðK½Lꢃ Þ Þꢃ to get a series of equation with
3
for [Pt(bpy)(Bu-dtc)]NO
3
and the inset for [Pd(bpy)
0
(
3
. These plots are curvilinear concave downwards, sug-
39
l
f
, m
n
n
plex (1.4 mmol/L) was added to each cell. After 3 min, the absorp-
tion was recorded at 258 nm for DNA and at 640 nm to eliminate
the interference of turbidity. Addition of metal complex to both
tion [
m
f
f
unknown parameters n, K, and g. Using Eureka software, the theo-
8
6
4
2
00
00
00
00
0
0.25
0
.4
1
00
0.2
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
0.35
0.3
2
3
7 °C
7 °C
0.15
ν
27 °C
37 °C
0
0
0
.1
.05
0.25
0
.05
0.1
ν
0.15
0.2
-13
-11
Ln[L]f
-9
-7
ν
0.2
0.15
0.1
2
7 °C
7 °C
2
7 °C
7 °C
3
3
0
0
.05
-8
-7
-6
Ln[L]f
-5
-4
0
0.1
0.2
0.3
0.4
ν
Figure 3. Scatchard plots for binding of [Pt(bpy)(Bu-dtc)]NO
3
with DNA. The inset
Figure 4. Binding isotherm plots for [Pt(bpy)(Bu-dtc)]NO
DNA. Inset, for [Pd(bpy)(Bu-dtc)]NO
3
in the interaction with
is Scatchard plots for binding of [Pd(bpy)(Bu-dtc)]NO with DNA.
3
3
.

9
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H. Mansouri-Torshizi et al. / Bioorg. Med. Chem. 16 (2008) 9616–9625
5
4
3
2
1
00
00
00
00
00
0
Furthermore, some thermodynamic parameters found in the
5
4
3
2
1
00
00
00
00
00
0
process of DNA denaturation are discussed as follows: Using the
DNA denaturation plots given in Figure 6 and Pace method,43 the
values of K, unfolding equilibrium constant and
energy of DNA at two temperatures of 27 and 37 °C in the presence
of [Pt(bpy)(Bu-dtc)]NO and [Pd(bpy)(Bu-dtc)]NO have been cal-
culated. A straight line is obtained when the values of G° are plot-
DG°, unfolding free
3
3
D
0
0.02
0.04
ted versus the concentrations of each metal complex in the
transition region at 27 and 37 °C. These plots are shown in Figure 7
for Pt(II) and the inset for Pd(II) systems. The m, slope of these plots
[
L]f (mM)
(
a measure of the metal complex ability to denature DNA) and the
ꢄ
ðH
intercept on ordinate,
DG
, (conformational stability of DNA in
OÞ
2
the absence of metal complex) are summarized in Table 4. The val-
ues of m for Pd(II) complex are higher than that of Pt(II) complex,
which indicate the higher ability of Pd(II) to denature DNA. These
m values are similar to that of Pd(II) complex as well as surfactant
1
6
reported earlier. As we know, the higher the value of
ger the conformational stability of DNA. However, the values of
G° (see Table 4) are decreased by increasing the temperature
DG°, the lar-
-
100
D
0
0.01
0.02
0.03
0.04
0.05
for both complexes. This is as expected because in general, most
[
L] (mM)
41
f
of the macromolecules are less stable at higher temperature. An-
other important thermodynamic parameter found is the molar en-
Figure 5. Molar enthalpies of binding in the interaction between DNA and
Pt(bpy)(Bu-dtc)]NO (inset, Pd(bpy)(Bu-dtc)]NO ) versus free concentrations of
complexes at pH 7.0 and 27 °C.
thalpy of DNA denaturation in the absence of metal complexes,
[
3
3
ꢄ
ðH
that is,
DH
. For this, we calculated the molar enthalpy of
2
OÞ
DNA denaturation in the presence of each metal complex,
ꢄ
DH
conformation or
DH
, in the range of the two temperatures
denaturation
4
4
cells was continued until no further changes in the absorption
readings were observed. The profiles of denaturation of DNA by
using Gibbs–Helmholtz equation. On plotting the values of these
enthalpies versus the concentrations of each metal complex,
straight lines will be obtained which are shown in Figure 8 for
[Pt(bpy)(Bu-dtc)]NO3 and the inset for [Pd(bpy)(Bu-dtc)]NO3.
[
3 3
Pt(bpy)(Bu-dtc)]NO and [Pd(bpy)(Bu-dtc)]NO are shown in Fig-
ure 6 at the two temperatures of 27 and 37 °C. The concentration
of metal complexes in the midpoint of transition, [L]1/2, for Pt(II)
complex at 27 °C is 0.145 and at 37 °C is 0.137 mmol/L and for
Pd(II) complex at 27 °C is 0.093 and at 37 °C is 0.085 mmol/L. The
improving effect of the temperature, leading to the decrease in
Intrapolation of these lines (intercept on ordinate i.e., absence of
ꢄ
ðH
metal complex) gives the values of
DH
(see Table 4). These
2
OÞ
plots show that in the range of 27–37 °C the changes in the enthal-
pies in the presence of Pd(II) complex is descending, while that of
Pt(II) is ascending. These observations indicate that by increasing
the concentration of Pd(II) complex, the stability of DNA is de-
creased while in the case of Pt(II) the opposite trend is observed
[
0
L]1/2 from 0.145 to 0.137 for Pt(II) system and from 0.093 to
.085 for Pd(II) system, indicates that the increase in the tempera-
ture lowers the stability of the DNA against denaturation caused by
these complexes. The important observation of this work is the low
which may be due to higher tendency of interaction of Pd(II) than
values of [L]1/2 for these complexes,4
0–42
that is, both complexes (in
ꢄ
Pt(II) complexes with DNA. In addition, the entropy (
DS
) of
ðH
2
OÞ
particular Pd(II) complex) can denature DNA at very low concen-
trations. Thus, if these complexes are used as anti-tumor agents,
low doses will be needed, this may have fewer side effects.
DNA unfolding by Pt(II) and Pd(II) complexes have been calculated
using equation G = S, and the data are given in Table 4.
D
D
H ꢁ T
D
These data show that the metal–DNA complex is more disordered
0
0
0
0
.4
.3
.2
.1
0
6
0
0
0
0
0
0
0
0
0
.9
.8
.7
.6
.5
.4
.3
.2
.1
0
27 °C
1
4
4
2
3
7 °C
27 °C
7 °C
0
3
-2
10
6
-
4
-6
0
0.1
[L] (mM)
0.2
t
0
0.1
0.2
[L]t (mM)
2
.
.
27 °C
37 °C
.
27 °C
-
-
2
.
37 °C
6
0
0.1
0.2
L] (mM)
0.3
0.4
0.09
0.11
0.13
0.15
[L] (mM)
0.17
0.19
0.2
[
t
t
Figure 6. The changes of absorbance of DNA at kmax = 258 nm due to increasing the
total concentration of [Pt(bpy)(Bu-dtc)]NO and the inset, [Pd(bpy)(Bu-dtc)]NO
L] , at constant temperature of 27 and 37 °C.
Figure 7. The molar Gibbs free energies plots of unfolding (
the presence of [Pt(bpy)(Bu-dtc)]NO
(Bu-dtc)]NO
D
G° vs [L]
t
) of DNA in
3
3
,
3
. Inset, in the presence of [Pd(bpy)
[
t
3
.

H. Mansouri-Torshizi et al. / Bioorg. Med. Chem. 16 (2008) 9616–9625
9621
Table 4
Thermodynamic parameters of DNA denaturation by platinum (II) and palladium (II) complexes
ꢄ
ðH
ꢄ
ðH
ꢄ
ðH
ma (kJ/mol))mmol/L)ꢁ1
DG
b
(kJ/molK)
DH
c
(kJ/mol)
DS
d
(kJ/mol)
OÞ
Compound
Temperature (°C)
2
OÞ
2
OÞ
2
[
Pt(bpy)(Bu-dtc)]NO
3
27
96.1
93.4
13.61
12.98
5.89
ꢀ0
0.073
3
7
[
Pd(bpy)(Bu-dtc)]NO
3
27
72.12
89.9
7.47
6.74
29.36
3
7
a
Measure of the metal complex ability to denature DNA.
b
c
Conformational stability of DNA in the absence of metal complex.
The heat needed for DNA denaturation in the absence of metal complex.
The entropy of DNA denaturation by metal complex.
d
5
3
1
0
0
0
eluted fraction of 2 mL was monitored at 258 nm. The gel chro-
matogram obtained is shown in Figure 9C. This indicates that the
highly polymerized calf thymus DNA has fragments with approxi-
mately similar molecular weights.
1
1
1
2.5
1.5
0.5
-
10
-30
50
The binding between DNA and metal complexes was also stud-
ied by precipitating the DNA from the interacted DNA–metal com-
plexes with absolute ethanol. In this experiment, the precipitated
DNA was separated out and washed several times with the alcohol.
This precipitate was redissolved in Tris–HCl buffer, and the solu-
tion was monitored spectrophotometrically for DNA at 258 nm,
Pt(II) complex at 321 nm, and Pd(II) complex at 317 nm. The pres-
ence of DNA–metal complex in this solution, as observed by spec-
tral method, suggests that the bonding of these metal complexes
with DNA is strong enough and it does not break readily, and it
is responsible for stabilizing the above DNA–metal complexes. Be-
cause, the interaction between DNA and involved metal complex
was very weak, the DNA should be precipitated separately and
-
9
8
7
6
5
.5
.5
.5
.5
.5
0
0.05
0.1
0.15
[L]t (mM)
0
0.05
0.1
L]t (mM)
0.15
0.2
45
all free metal complexes would remain in the supernatant.
[
2
.3.4. Intercalation studies using fluorescence method
Fluorescence titration of solutions containing the DNA and ethi-
Figure 8. Plots of the molar enthalpies of DNA denaturation in the interaction with
Pt(bpy)(Bu-dtc)]NO and the inset with [Pd(bpy)(Bu-dtc)]NO complexes in the
range of 27–37 °C.
[
3
3
dium bromide with Pt(II) or Pd(II) complexes has been investi-
gated. It is known that the fluorescence intensity of ethidium
bromide grows when it goes from a polar to a nonpolar medium
due to the lowering of the inter-system crossing lifetimes.46 Ethi-
dium bromide is a dye whose fluorescence is quenched in water,
but strongly enhanced when it intercalates within DNA duplexes.47
The fluorescence emission spectra of the intercalated ethidium
with increasing concentrations of Pd(II) and Pt(II) complexes are
shown in Figure 10. Figure 10 shows a significant reduction of
the ethidium emission intensity by adding different concentrations
of Pd(II) and Pt(II) complexes (a: 0.00, b: 0.05, c: 0.1, and d:
0.15 mM) at 27 °C. Similar observations were made when the
experiments were carried out at 37 °C. It can then be concluded
that the fluorescence intensity of DNA-intercalated ethidium is
quenched, when ethidium is removed from the duplexes by the ac-
tion of Pd(II) and Pt(II) complexes and it is released into buffer.
These results suggest that the above metal complexes presumably
than that of native DNA, because the entropy changes are positive
and the extent of disorder in Pd(II)–DNA complex is more than that
of Pt(II)–DNA complex (see Table 4). This again shows that ability
of palladium complex in the denaturation of DNA is more than that
of the platinum complex.
2
.3.3. Evaluation of binding modes
The solution of each interacted DNA–metal complex was passed
through a Sephadex G-25 column equilibrated with Tris–HCl buf-
fer. Elution was done with buffer and each fraction of the column
was monitored spectrophotometrically at 321 and 258 nm for
Pt(II)–DNA system and 317 and 258 nm for Pd(II)–DNA system.
The gel chromatograms obtained from these experiments are given
inFigure 9A and B. These results show that the two peaks obtained
at two wavelengths were not clearly resolved, which indicate that
metal complexes have not separated from DNA and their bindings
with DNA are strong enough and do not break readily. This type of
binding of metal complexes to DNA is strictly true for kinetically
inert platinum(II)–DNA complex. However, in the case of palla-
dium(II)–DNA system, for both aforementioned wavelengths two
peaks were observed none of which were resolved. This indicates
that in the presence of palladium(II) complex, DNA partially breaks
into two fragments, one with higher and the other one with lower
molecular weight. Surprisingly, the amount of palladium(II) metal
complex bound to the fragment with lower molecular weight is
higher than the amount bound to the fragment with higher molec-
ular weight as indicated from the absorption readings at 317 nm.
To confirm the breaking of DNA by this metal complex, a solution
of DNA alone was passed through the same column and each
0
intercalate into DNA through the planar 2,2 -bipyridine ligand
present in their structures. Similar observations have been made
in the interaction of [Pt(bpy)(ddtc)]NO , where ddtc is diethyldi-
thiocarbamate, with calf thymus DNA.
3
1
2
2.3.5. Circular dichroism
The CD spectral technique is useful in monitoring the conforma-
tional variations of DNA in solution. Thus, CD spectra of calf thy-
mus DNA incubated with platinum or palladium complexes at
several ratios were recorded. The observed CD spectrum of
DNA consists of a positive band at 265 nm due to base stacking
and a negative band at 247 nm due to helicity, which are character-
istics of calf thymus DNA (Fig. 11a). When the complexes are free
in the solution, they do not have any CD spectrum; whereas they
have an induced CD spectrum as interact with DNA. When our
3
8

9
622
H. Mansouri-Torshizi et al. / Bioorg. Med. Chem. 16 (2008) 9616–9625
crocin and crocetin with calf thymus DNA.41 Moreover, in the case
0
0
0
0
0
0
0
.7
.6
.5
.4
.3
.2
.1
0
A
of Pd complex a further decrease in the positive and negative bands
was observed at concentrations higher than [L]1/2 (Fig. 11c, inset).
This indicates that conformational changes in the structure of DNA
in the presence of Pd complex must be different from that of Pt
complex.
Abs in 258 nm
Abs in 317 nm
3
. Conclusion
Two water soluble Pt(II) and Pd(II) complexes have been syn-
thesized and characterized. They exhibited potent cytotoxic prop-
erties against chronic myelogenous leukemia cell line, K562. The
cells showed different sensitivities to complexes. Cc50 values of
Pt(II) complex are slightly lower than those of Pd(II) complex
and both are much lower than those of cisplatin. Both complexes
have been interacted with calf thymus DNA. They cooperatively
bind to DNA presumably through intercalations. They unexpect-
edly denature DNA at very low concentrations. Determination of
several binding and thermodynamic parameters has also been at-
tempted. Palladium complex breaks the DNA into two unequal
fragments and binds stronger to the lighter fragment than the
heavier one.
0
5
10
15
Effluent (ml)
0
0
0
0
0
0
0
0
0
.9
.8
.7
.6
.5
.4
.3
.2
.1
0
B
Abs in 258 nm
Abs in 321 nm
4
4
. Experimental
.1. Materials and methods
0
Potassium tetrachloroplatinate, 2,2 -bipyridine, highly poly-
merized calf thymus DNA sodium salt and Tris–HCl buffer were ob-
tained from Merck (Germany). Palladium(II) chloride anhydrous
was bought from Fluka (Switzerland). Butylamine and carbon
0
0.1
5
10
15
20
25
-
Effluent (ml)
disulfide were purchased from Aldrich (England). [Pt(bpy)Cl
2
]
and [Pd(bpy)Cl ] were prepared by the procedure described in
2
4
8
0
0
0
0
0
0
0
0
.8
.7
.6
.5
.4
.3
.2
.1
0
the literature. Solvents used were of reagent grade and purified
before being used by the standard methods. Other chemicals used
were of analytical reagent or of higher purity grade.
C
Infrared spectra of the metal complexes were recorded on a JAS-
CO-460 Plus FT-IR spectrophotometer in the range of 4000 to
ꢁ
1
4
00 cm in KBr pellets. Microchemical analysis of carbon, hydro-
gen, and nitrogen for the complexes was carried out on Herause
1
CHNO-RAPID elemental analyzer. H NMR spectra were recorded
on a Brucker DRX-500 Avance spectrometer at 500 MHz in
DMSO-d using tetramethylsilane as internal reference. Electronic
6
Abs in 258 nm
absorption spectra of the title metal complexes were measured on
a JASCO UV/VIS-7850 recording spectrophotometer. Melting points
were measured on a Unimelt capillary melting point apparatus
and here reported uncorrectedly. Conductivity measurements of
the above platinum and palladium complexes were carried out
on a Systronics conductivity bridge 305, using a conductivity cell
of cell constant 1.0. The doubly distilled water was used as solvent.
Fluorescence intensity measurements were carried out on a Hitachi
spectrofluorimeter model MPF-4. Circular dichroism spectra were
recorded on an Aviv Spectropolarimeter model 215.
0
5
10
15
20
Effluent (ml)
Figure 9. Gel chromatograms of (A) [Pt(bpy)(Bu-dtc)]NO
Pd(bpy)(Bu-dtc)]NO
3
–DNA complex, (B)
[
2
0
3
–DNA complex and (C) DNA alone, obtained on Sephadex G-
5 column, equilibrated with 10 mmol/L Tris–HCl buffer of pH 7.0 in the presence of
.01 mol/L sodium chloride.
4.2. Synthesis of ligand and metal complexes
4
.2.1. Synthesis of Bu-dtcNa
This ligand was synthesized by a modified literature method49
:
compounds were incubated with DNA, the CD spectra displayed
changes of both positive and negative bands (Fig. 11b and c). At
concentrations lower than [L]1/2 (see Section 2.3.2), the complexes
may induce certain conformational changes in DNA as indicated by
significant decrease in the intensities of positive and negative
butylamine (5 mL, 50 mmol) and sodium hydroxide (2.0 g,
0 mmol) were added to 50 mL acetone and stirred for one hour
5
at 0 °C in an ice bath. Carbon disulfide (5 mL, excess) was added
dropwise while vigorous stirring. The curdy yellow solution so ob-
tained was stirred for another 4 h at 0 °C and kept overnight at
room temperature. It was then filtered and 100 mL diethylether
was added to the filtrate and placed in refrigerator overnight.
bands (Fig. 11b). However, at concentrations higher than [L]1/2
the precipitate formed and spectra collapsed completely (Fig.
1c). Similar observations have been made in the interaction of
,
1

H. Mansouri-Torshizi et al. / Bioorg. Med. Chem. 16 (2008) 9616–9625
9623
A
400
B
400
a
a
3
3
2
50
00
50
3
3
2
2
1
1
50
00
50
00
50
00
b
c
d
b
c
d
200
150
100
50
5
0
0
0
520
570
620
Wavelenght (nm)
670
720
5
45
595
645
695
Wavelenght (nm)
Figure 10. Fluorescence emission spectra of interacted ethidium–DNA in the absence (a) and presence of different concentrations of Pt(II) complex (A) and Pd(II) complex (B):
.05 mM (b); 0.1 mM (c); 0.15 mM (d) at 27 °C.
0
was repeated with the second portion of acetone to remove most
of the impurities. The orange powder so obtained was dissolved
in 10 mL methanol/acetonitrile (1:1 v/v) mixture and filtered. Dif-
fusion of ether into this filtrate gave brown needle-shaped crys-
tals after three days. The crystals were isolated by filtration,
washed with 15 mL acetone, and dried at 40 °C. Yield: 0.330 g
(59%) and decomposed at 235 °C. Anal. Calcd for C15H N O S Pt
18 4 3 2
(561): C, 35.09; H, 3.21; N, 9.98%. Found: C, 35.03; H, 3.22; N,
10.00%.
1
0
8
6
4
2
0
9
4
a
b
c
-1
200
250
Wavelenght (nm)
300
a
4
.2.3. Synthesis of [Pd(bpy)(Bu-dtc)]NO
This complex was prepared by a similar method to that of
Pt(bpy)(Bu-dtc)]NO . Yield: 0.273 g (58%) and decomposed at
170 °C. Anal. Calcd For C15 Pd (472): C, 38.14; H, 3.81;
3
c
b
[
3
18 4 3 2
H N O S
2
00
220
240
260
280
300
320
N, 11.86%. Found: C, 37.99; H, 3.80; N, 11.80%.
-2
Wavelenght (nm)
4.3. Cytotoxic studies
Figure 11. CD spectra of DNA adduct with compound. Plot for titration of DNA with
the Pt(II) complex and inset for Pd(II) complex at 27 °C. (a) Free DNA (120 M); (b)
in the presence of 100 M complex and (c) in the presence of 200 M complex.
4
.3.1. Cell culture
In RPMI medium, chronic myelogenous leukemia cells were
l
l
l
grown. This medium was supplemented with
L-glutamine
(
2 mM), streptomycin and penicillin (5 g/mL), and 10% heat-inac-
l
The resulting white precipitate of crude product was filtered off
and vacuum dried. Recrystallization was carried out by stirring
the crude product in 35 mL acetone and filtering the undissolved
particles out. Thirty microliters dichloromethane was added to
the filtrate and then left in a refrigerator overnight. The desired
product was collected by filtration as white needle-like crystals
and washed with small amount of dichloromethane and vacuum
dried. Yield was 6.84 g (80%) with a melting point of 73 °C. Anal.
2
tivated fetal calf serum, at 37 °C under a 5% CO /95% air
atmosphere.
4.3.2. Cell proliferation assay
The above Pt(II) and Pd(II) complexes inhibit the growth of
chronic myelogenous leukemia cell line, K562. This growth inhibi-
tion was measured by means of MTT assay. The cleavage and con-
version of the soluble yellowish MTT to the insoluble purple
formazan by active mitochondrial dehydrogenase of living cells
has been used to develop an assay system alternative to other as-
says for measurement of cell proliferation. Harvested cells were
5 2
Calcd for C H10NS Na (171.12): C, 35.09; H, 5.85; N, 8.19%. Found:
C, 35.02; H, 5.84; N, 8.18%.
4
4
.2.2. Synthesis of [Pt(bpy)(Bu-dtc)]NO
Pt(bpy)Cl ] (0.422 g, 1 mmol) was suspended in 80 mL doubly
distilled water, and (0.34 g, 2 mmol) of AgNO was added to it
3
seeded into 96-well plate (2 ꢂ 10 cell/mL) with varying concen-
[
2
trations of the sterilized drugs (0–250 lM) and incubated for
3
24 h. At the end of four hours incubations 25
lL of MTT solution
with constant stirring. This reaction mixture was heated with
stirring under dark for 6 h at 60 °C and then for 16 h at room
temperature (30 °C). The AgCl precipitate was filtered through
Whatman 42 filter paper. To the clear yellow filtrate containing
(5 mg/mL in PBS) was added to each well containing, fresh and cul-
3
8
tured medium. At the end, the insoluble formazan produced was
dissolved in solution containing 10% SDS and 50% DMF (Left for 2 h
at 37 °C in dark conditions), and optical density (OD) was read
against reagent blank with multi well scanning spectrophotometer
(ELISA reader, Model Expert 96, Asys Hitchech, Austria) at a wave-
length of 570 nm. Absorbance is a function of concentration of con-
verted dye. The OD value of study groups was divided by the OD
value of untreated control and presented as percentage of control
(as 100%).
[
2 2 3 2
Pt(bpy)(H O) ](NO ) , a solution of 0.171 g, 1 mmol Butyldithioc-
arbamate sodium salt in 15 mL water was slowly added. Stirring
was continued at ꢀ50 °C for another 6 h and filtered. The clear
yellowish orange filtrate was evaporated at 35–40 °C to complete
dryness. The precipitate obtained was stirred with 15 mL acetone
to get fine powder. The acetone was decanted and the process

9
624
H. Mansouri-Torshizi et al. / Bioorg. Med. Chem. 16 (2008) 9616–9625
4
.3.3. Statistical analysis
Results were analyzed for statistical significance using two
tion containing 4 mg/mL) in 1–1.5 mL of the above-mentioned
Tris–HCl buffer of pH 7.0. To this interacted solution, an excess of
absolute ethanol was added. The precipitated DNA was separated
and washed with alcohol. This precipitate was redissolved in the
same buffer, and the solution was monitored spectrophotometri-
cally for DNA at 258 nm and Pt(II) or Pd(II) complexes at their
tailed Student’s t-test. Changes were considered significant at
p < 0.05.
4
.4. Binding studies
kmax (nm).
Calf thymus DNA alters the absorption spectra of [Pt(bpy)(Bu-
complexes.5
0,51
The stock solu-
4.4.5. Fluorescence measurements
dtc)]NO
3
and [Pd(bpy)(Bu-dtc)]NO
3
tions of Pt(II) and Pd(II) complexes (1.4 mmol/L) were made in the
above-mentioned Tris–HCl buffer of pH 7.0 medium containing
The fluorescence of ethidium bromide (EthBr) is greatly en-
hanced on its intercalation between the base pairs of DNA.46 Ethi-
dium bromide displacement assay was performed as reported in
1
3
0 mmol/L sodium chloride by gentle stirring and heating at
5 °C, while that of DNA (4 mg/mL) at 4 °C until homogenous.
4
2
the literature. At first, DNA (60 lM) was added to 2 lM aqueous
The metal complex solutions with and without DNA were incu-
bated at 27 and 37 °C. Then, the spectrophotometric readings at
ethidium bromide solution and maximum quantum yield for ethi-
dium bromide was achieved at 471 nm, so we selected this wave-
length as excitation radiation for all samples at different
temperatures (27 °C and 37 °C) in the range of 540–700 nm. To this
solution (containing ethidium bromide and DNA), different con-
centrations of the Pd(II) or Pt(II) complex were added (0.05, 0.1
and 0.15 mM). Measurements were done by applying a 1-cm path
length fluorescence cuvette. The fluorescence intensities of the
Pd(II) and Pt(II) complexes at the highest denaturant concentration
at 471 nm excitation wavelength have been checked, and the emis-
sion intensities of these compounds were very small and
negligible.
kmax (nm) of complexes, where DNA has no absorption, were mea-
sured. Using trial and error method, the incubation time for solu-
tions of DNA–metal complexes at 27 and 37 °C was found to be
6
.5 h. No further changes were observed in the absorbance reading
after longer incubation. The concentration of DNA was found out
based on determination of phosphate (P). Millimolar extinction
coefficient of native DNA solution at E258 (e258) based on DNA P
3
52
was 6.6 ꢂ 10 . Methods for DNA-binding studies of above com-
plexes are described as follows.
4
.4.1. Determination of binding parameters
In the interaction of the metal complexes with DNA, the proce-
4.4.6. CD spectropolarimetry
dures followed to determine n, K, and g, where n is Hill coefficient,
g is the number of binding sites per 1000 nucleotides of DNA, and K
is apparent binding constant, were similar to what was reported
Circular dichroism spectra showed changes in the structure of
DNA, which were monitored in the region (200–320 nm) using 1-
4
6
cm path length cells. The DNA concentration in the experiments
was 120 M. Induced CD spectra resulting from the interaction of
the Pd(II) or Pt(II) complex (100 and 200 M) with DNA at the tem-
3
9
earlier. Also, the other thermodynamic-binding parameters: mo-
l
ꢄ
b
lar Gibbs free energy of binding ð
D
G Þ, molar enthalpy of binding
l
ꢄ
b
ꢄ
b
ðD
H Þ, and molar entropy of binding ð
D
S Þ were determined
peratures of 27 °C and 37 °C were obtained by subtracting the CD
spectrum of the native DNA and mixture of DNA–Pd(II) or –Pt(II)
complex from the CD spectrum of the buffer and spectrum of buf-
fer–Pd(II) or –Pt(II) complex solutions.
16
according to reported method.
4
.4.2. DNA-denaturation studies
Denaturation of DNA was carried out by looking at the changes
in the UV absorption spectrum of DNA solution in 258 nm upon
Acknowledgments
16,41
addition of Pt(II) or Pd(II) complexes.
centration of each metal complex at midpoint of transition, [L]1/2
was determined. Also, thermodynamic parameters such as:
In these studies, the con-
,
Financial assistance from the Research Council of University of
Sistan and Baluchestan and of University of Tehran is gratefully
acknowledged.
ꢄ
ðH
D
G
, conformational stability of DNA in the absence of metal
OÞ
ꢄ
2
complex;
sence of metal complex;
DH
, the heat needed for DNA denaturation in the ab-
OÞ
ꢄ
ðH
2
DS
, the entropy of DNA denaturation
ðH
2
OÞ
References and notes
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